Distribution and Methylation of CpG dinucleotide sequences. DNA methylation in mammalian cells is required for embryonic development, X-chromosome inactivation and genomic imprinting, and involves the addition of a methyl group to the C-5 position of cytosine, predominantly in a 5′-CpG-3′ (CpG dinucleotide) sequence context (reviewed in (Bird, A., Genes Dev., 16:6-21, 2002). This is accomplished by the activities of one or more DNA methyltransferases (DNMTs) that use S-adenosylmethionine (AdoMet) as a cofactor. CpG dinucleotides are underrepresented in the human genome by a factor of 5, due to the spontaneous deamination of 5-methylcytosine residues, resulting in C-to-T transition mutations at CpG dinucleotides (Laird, P. W., Natl. Rev. Cancer 3:253-266, 2003). However, there are regions of the genome, termed ‘CpG islands’ that have retained their expected CpG content (Gardener-Garden & Fommer, J. Mol. Biol., 196:261-282, 1987; and Takai and Jones, Proc. Natl. Acad. Sci. USA 99:3740-3745, 2002). Most CpG Islands overlap the 5′ end of gene regions including promoters and are typically unmethylated in normal somatic tissues (see, e.g., Takai and Jones, Proc. Natl. Acad. Sci. USA 99:3740-3745, 2002; Jones and Baylin, Natl. Rev. Genet. 3:415-428, 2002). However, only 40% of promoter regions are associated with CpG islands (Id). The unique and repeated sequences in the remainder of the genome are often highly methylated at their CpG sites in somatic tissues (Ehrlich, Oncogene 21:5400-5413, 2002).
Aberrant CpG methylation in cancer. CpG dinucleotides are often aberrantly methylated in human cancers to give regional hypermethylation at some CpG islands despite an overall reduction in 5-methylcytosine in the DNA (despite, global CpG hypomethylation) (Gama-Sosa et al., Nucl. Acids. Res. 11:6883-6894, 1983; Feinberg and Vogelstein, Nature 301:89-92, 1983; Feinberg et al., Cancer Res. 48:1159-1161, 1988). The frequent hypomethylation of repetitive elements in diverse human cancers may largely account for the global hypomethylation commonly seen in human cancers (Ehrlich, 2002, supra).
Types, distribution and methylation of Repetitive DNA sequence elements. Repetitive elements comprise approximately 45% of the human genome (see e.g., Lander et al., Nature 409, 860-921, 2001; Jordan et al., Trends Genet. 19:68-72, 2003) and consist of interspersed repeats derived from non-autonomous or autonomous transposable elements (reviewed in Weiner, Curr. Opin. Cell Biol. 14:343-350, 2002; Deininger et al., Curr. Opin. Genet. Dev. 13:651-658, 2003; and Prak & Kazazian, Natl. Rev. Genet. 1:134-144, 2000) and tandem repeats of simple sequences (satellite DNA) or complex sequences. The most plentiful short interspersed nucleotide element (SINE) in human DNA is the Alu repeat, a 282 bp non-LTR (non-Long Terminal Repeat) DNA sequence, which comprises 10% of the human genome and is present in about 1 million copies per haploid genome (Weiner, 2002, supra). Other abundant non-LTR sequences are long interspersed nucleotide elements (LINEs) of up to 6 kb that comprise nearly 20% of the human genome (reviewed in Ehrlich, 2002, supra; and in Deininger, 2003, supra).
Long interspersed nuclear elements (LINEs) are autonomous retrotransposons that are DNA sequences up to about 6 kb (e.g., of about 4-6 kb) in length that comprise nearly 20% of the human genome (reviewed in Ehrlich, Supra; Deininger, Supra). LINE-1 elements are present at over 500,000 copies in the human genome, however, only 3,000-4,000 are full length and 30-100 are active retrotransposons (Id).
LINE-1 elements are usually methylated in somatic tissues, and LINE-1 hypomethylation is a common characteristic of human cancers (Kimura et al., Int. J. Cancer 104:568-578, 2003; Florl et al., Br. J. Cancer 91:985-994, 2004; Chalitchagom et al., Oncogene 23:8841-8846, 2004; and Yang et al., Nucleic Acids Res., 32:e38, 2004). Moreover, Alu sequences are also normally methylated in somatic tissues (Gama-Sosa et al., Nucleic Acids Res., 11:3087-3095, 1983; Schmid, C. W., Nucleic Acids Res., 19:5613-5617, 1991; Kochanek et al, EMBO J. 12:1141-1151, 1993) and are thought to become hypomethylated in human cancer cells (Yang, 2004, supra). However, not all Alus are hypomethylated in human cancers. Alu sequences located upstream of the CDKN2A promoter were found to be hypermethylated in cancer cell lines (Weisenberger et al., Mol. Cancer Res., 2:62-72, 2004), and an Alu sequence located in intron 6 of TP53 showed extensive methylation in normal and cancer cells (Id).
While LINEs and SINEs are interspersed throughout the genome, satellite DNA is largely confined to the centromeres or centromere-adjacent (juxtacentromeric) heterochromatin and to the large region of heterochromatin on the long arm of the Y chromosome. Satellite α (Satα) repeats are composed of 170-bp sequences and represent the main DNA component of every human centromere (Lee et al., Human Genet. 100:291-304, 1997). Satellite 2 (Sat2) DNA sequences are found predominantly in juxtacentromeric heterochromatin of certain human chromosomes, and are most abundant in the long juxtacentromeric heterochromatin region of chromosome (Chr) 1. Sat2 sequences are composed of variants of two tandem repeats of ATTCCATTCG (SEQ ID NO:27) followed by one or two copies of ATG (Jeanpierre, M., Ann. Genet. 37:163-171, 1994). Both Chr1 Satα: and Chr1 Sat2 sequences as well as Satα repeats present throughout all the centromeres are highly methylated in normal postnatal tissues, hypomethylated in sperm, and often hypomethylated in various cancers (Narayan et al., Intl. J. Cancer 77:833-838, 1998; Qu et al., Mutat. Res. 423:91-101, 1999; Qu, et al., Cancer Genet. Cytoget. 109:34-39, 1999; and Ehrlich et al., Cancer Genet. Cytogenet. 141:97-105, 2003). Additionally, Sat2 sequences on Chr1 and Chr16 are also hypomethylated in the ICF (immunodeficiency, centromeric region instability and facial abnormalities) syndrome, which usually involves mutations in DNMT3B ((Xu et al., Nature 402:187-191, 1999; Hansen et al., Proc. Natl. Acad. Sci. USA 96:14412-14417, 1999).
Prior art methods for determining repetitive element methylation. Although repetitive element DNA is abundant in the human genome, previous methods to determine repetitive element methylation have been based on restriction enzyme digestion and Southern blot analyses, which require large amounts of high-molecular weight genomic DNA (Gama-Sosa, Supra; Qu et al., Mutat. Res. 423:91-101, 1999; Ehrlich et al., Cancer Genet. Cytogenet., 141:97-105, 2003; Ehrlich et al., Nucleic Acids Res., 10:2709-2727, 1982; and Widschwendter et al., Cancer Res. 64:4472-2280, 2004 (A)). Moreover, accurate global genomic 5-methylcytosine content is often determined by high performance liquid chromatography (BPLC) (Id), which, although highly quantitative and reproducible, also requires large amounts of high-quality genomic DNA and is not suitable for high-throughput analyses. Alu or LINE-1 methylation levels were recently obtained by COBRA (Combined Bisulfite Restriction Analysis) and pyrosequencing of bisulfite converted DNA (Yang et al., Nucleic Acids Res., 32:e38, 2004). Although these quantitative methods represent improvements in determining repetitive element DNA methylation levels, they have not been correlated with global hypomethylation (only with demethylation in particular cell lines as a consequence of treatment with 5-aza-2′deoxycytidine (DAC)), and both require post-PCR manipulation, are labor-intensive, and therefore may not be suitable for high-throughput analyses.
There is a pronounced need in the art for assays having utility to measure global DNA methylation using relatively small amounts of genomic DNA, and that are suitable for high-throughput analyses.
There is a pronounced need in the art for the development control reactions (e.g., an Alu-based control reaction) that are both more sensitive for detecting minute quantities of DNA, and that are far less sensitive/subject to local cancer-associated genetic alterantions (e.g., to detect input DNA levels from samples with chromosomal instability, compared to single-copy control genes that we have traditionally used.
There is a pronounced need in the art for the development of a novel strategy that allows the discrimination between evolutionary- and bisulfite-based deamination of a particular DNA sequence, and for application of this strategy in the design of a bisulfite-specific real-time PCR reaction towards, for example, the Alu consensus sequence having utility for detecting small amounts of DNA (e.g., DNA from formalin-fixed, paraffin-embedded tissues or free tumor DNA in plasma or serum).
There is a pronounced need in the art for the testing and validation (e.g., on a panel of normal and tumor DNA samples for which HPLC-based global DNA methylation measurements has been performed) of high-throughput assays (e.g., MethyLight™) for determining the methylation status of Alu, LINE-1, and chromosome 1 centormeric satα and juxtacentromeric sat2 repeats.
There is a pronounced need in the art for the development of surrogate markers for estimating global DNA methylation levels.